Staphylococcus pseudintermedius exfoliative toxin EXI selectively digests canine desmoglein 1 and causes subcorneal clefts in canine epidermis.

Staphylococcal exfoliative toxins are known to digest desmoglein (Dsg) 1, a desmosomal cell-cell adhesion molecule, thus causing intraepidermal splitting in human bullous impetigo, staphylococcal scalded skin syndrome and swine exudative epidermitis. Recently, a novel exfoliative toxin gene (exi), whose sequence shares significant homology with previously identified exfoliative toxins, was isolated from Staphylococcus pseudintermedius. Little is known about the pathogenic involvement of this toxin in canine pustular diseases such as impetigo. The aim of this study was to determine whether EXI, the product of the exi gene, digests canine Dsg1 and causes intraepidermal splitting in canine skin. An exi gene was isolated from chromosomal DNA of an S. pseudintermedius strain obtained from a pustule of a dog with impetigo, and was used to produce a recombinant EXI by Escherichia coli expression. When purified recombinant EXI was injected intradermally into normal dogs, it caused the development of vesicles or erosions with superficial epidermal splitting. In addition, the EXI abolished immunofluorescence for Dsg1, but not for Dsg3, at the injection sites. Moreover, the EXI directly degraded baculovirus-secreted recombinant extracellular domains of canine Dsg1, but not that of canine Dsg3, in vitro. The EXI also degraded mouse Dsg1α and swine Dsg1, but not human Dsg1, mouse Dsg1ß and Dsg1γ. Conversely, recombinant SIET, previously designated as S. intermedius exfoliative toxin, did not cause intraepidermal splitting or degradation of any Dsgs. These findings indicate that EXI has a proteolytic activity that digests canine Dsg1, and this characteristic might be involved in the pathogenesis of intraepidermal splitting in canine impetigo.

Epidemiological characteristics of Salmonella enterica serovar typhimurium from healthy pigs in Japan.

We characterized 53 Salmonella enterica ser. Typhimurium strains recovered from healthy pigs during 1998-1999 (n=12) and 2004-2005 (n=41) as to their carriage of DT104 spacer region, class 1 and 2 integrons, virulence genes (spvC, rck, and pefA), and XbaI- and BlnI-Pulsed-Field Gel Electrophoresis (PFGE) profiles. No DT104 strain was detected in 1998-1999, whereas 65.9% (27/41) of the strains in 2004-2005 were DT104 showing resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline, and cephaloridine (R-type ACSSuT+). Class 1 intergron-associated genes, aadA2 (1.0-kbp amplicon) and pse1 (1.2-kbp amplicon), were found in all the DT104 strains (27/27). One strain showing resistance to streptomycin, sulfonamides, tetracyclin, and trimethoprim (R-type SsuTTm) harbored another class 1 integron-associated gene (dhfrXII-orfF- aadA2) on 1.9-kbp amplicon. Virulence gene spvC was found in 92.5% (49/53) and rck and pefA were found in 88.7% (47/53) of the strains, whereas spvC, rck, and pefA were found in all the DT104 strains. Ser. Typhimurium strains were categorized into four clusters (X1, X2, X3, and X4a/X4b) by XbaI-PFGE, or into nine clusters (B1, B2, B3a/B3b, B4, B5, B6, B7, B8, B9a/B9b) by BlnI-PFGE analyses. DT104 strains were restricted into X2, or into B2, B3a/B3b, and B6 clusters, indicating that our multidrug-resistant DT104 strains from healthy pigs might have derived from at least three independent clones, with the most widespread clone being the cluster B6 strains isolated in Kanto, Tokai, Chugoku, and Kyushu regions.

Antimicrobial susceptibilities of exfoliative toxigenic and non-toxigenic Staphylococcus hyicus strains in Japan.

Staphylococcus hyicus isolates (n=207), including 150 exfolitative toxigenic and 57 non-toxigenic strains, were examined for their susceptibility to 13 antimicrobial agents by using the dehydrated 96-well MIC panel system. The frequency of their resistance to penicillin and ampicillin was 76.8% (159/207), followed by erythromycin (56%, 116/207), trimethoprim-sulfamethoxazole (28.5%, 59/207), chloramphenicol (24.2%, 50/207), kanamycin (19.8%, 41/207), and doxycycline (1.4%, 3/207). Resistance to chloramphenicol and trimethoprim-sulfamethoxazole was significantly higher in toxigenic strains than non-toxigenic strains (p<0.01), whereas kanamycin and erythromycin resistance was significantly higher in non-toxigenic strains (p<0.01 and <0.05, respectively). Resistance to two or more antimicrobials was observed in 85.5% (177/207) of total strains, with a significantly higher occurrence in toxigenic strains (89.3%, 134/150 vs. 75.4%, 43/57; p<0.05).

Nationwide molecular surveillance of exfoliative toxigenic Staphylococcus hyicus on pig farms across Japan.

We investigated the carriage of Staphylococcus hyicus and their exfoliative toxin genes (exhA, exhB, exhC, exhD, and shetb) among 424 pigs from 38 Japanese pig farms by PCR and characterized the isolates by 16S-23S intergenic spacer region polymorphism analysis. S. hyicus (n=207) were isolated from 17.9% of pigs and 72.5% of them were toxigenic. The isolation rate of toxigenic S. hyicus was four times higher in the pigs with exudative epidermitis than the healthy pigs (87.6% versus 19.6%; p<0.01). Among the toxins, exhA was the most prevalent, being detectable in 49.3% of toxin gene-positive strains. S. hyicus isolates were distributed into nine ITS-PCR types (a-i), with ITS-PCR types d, h, and e being predominant accounting for 70% (145/207) of total isolates or 76% (114/150) of toxin gene-positive strains. This study denotes high prevalence of toxigenic strains among S. hyicus circulating on pig farms in Japan.

High occurrence of multi-antimicrobial resistance in Staphylococcus intermedius isolates from healthy and diseased dogs and domesticated pigeons.

Staphylococcus intermedius isolates (n=106), including 44 dog isolates and 62 pigeon isolates, were examined for their susceptibility to ampicillin, cephalexin, erythromycin, gentamicin, kanamycin, lincomycin, norfloxacin, oxacillin, tetracycline, and vancomycin by standard disk-diffusion test. The frequencies of resistance to ampicillin, kanamycin, and tetracycline were significantly higher in dog isolates than pigeon isolates (95.5% vs. 0%, 31.8% vs. 0%, and 45.5% vs. 9.7%, respectively; P<0.01). Antimicrobial resistance patterns of dog isolates and pigeon isolates were categorized respectively into nine and five distinct profiles. Significantly higher occurrence of resistance to two or more antimicrobials was observed in dog isolates than pigeon isolates (54.5% vs. 12.9%; P<0.01) and also in domesticated pigeon isolates than non-domesticated pigeon isolates (53.3% vs. 0%; P<0.01).

Prevalence of virulence factors in Staphylococcus intermedius isolates from dogs and pigeons.

BACKGROUND: Staphylococcus intermedius has been isolated from healthy dogs and pigeons as well as diseased dogs. Similar to Staphylococcus aureus, S. intermedius is known to carry many virulence factors but most of these factors remain to be studied. In this study, we examined 106 S. intermedius isolates (44 dog isolates and 62 pigeon isolates) for their hemolytic activity, biofilm formation, protease activity, and clumping factor and protein A production. RESULTS: Forty-three dog isolates (97.7%) and all pigeon isolates were hemolytic on sheep RBCs with a mean hemolytic titer of 336.7 and 47.32, respectively, whereas 43 dog isolates (97.7%) and 11 pigeon isolates (17.7%) exhibited a significant difference in their hemolytic activity on rabbit RBCs with a mean hemolytic titer of 11.04 and 3.76, respectively (p < 0.0005). The mean biofilm formation activity for dog isolates was 0.49, which was significantly higher than that (0.33) for pigeon isolates (p < 0.0005). Twenty-four dog isolates (54.5%) and 11 pigeon isolates (17.7%) were protease positive. Twenty-four dog isolates (54.5%) were clumping factor- and protein A- positive. CONCLUSION: S. intermedius strains carrying the virulence factors examined in this study were more prevalent in dogs than pigeons.

Identification of first exfoliative toxin in Staphylococcus pseudintermedius.

Staphylococcus aureus, Staphylococcus hyicus, and Staphylococcus chromogenes are known to cause skin infections in human or animals by producing exfoliative toxins (ETs). Staphylococcus pseudintermedius can also cause canine pyoderma, but no exfoliative toxins or similar toxins have been reported. PCR with degenerate primers targeted to the conserved regions in ETA, ETB, and ETD from S. aureus and SHETB from S. hyicus, and subsequent chromosome walking identified a novel gene, designated as exi (exfoliative toxin of pseudintermedius) in S. pseudintermedius. EXI had significant homologies with the exfoliative toxins (43-68% identity), particularly with ETB (67.1%), ETD (67.9%), and SHETB (65.1%). Phylogenetic analysis showed close relation between EXI and ETB with a bootstrap value of 80%. Neonatal mice injected with the crude proteins from the culture supernatant or recombinant EXI showed gross blisters and/or characteristic skin exfoliation. The prevalence of exi assessed by dot-blot hybridization was 23.3% (10/43) in S. pseudintermedius isolates from canine pyoderma. The EXI reported herein is the first exfoliative toxin identified in S. pseudintermedius.

Prevalence and characterization of leukotoxin-producing Staphylococcus intermedius in Isolates from dogs and pigeons.

Staphylococcus intermedius isolates from dogs (n = 44) and pigeons (n = 62) were categorized into 12 types by intergenic ribosomal DNA spacer polymorphism analysis. All isolates from pigeons were lukS positive and all isolates from dogs were lukS and lukF positive by dot blot analysis. The mean leukotoxicity titer for dog isolates was at least 129-fold higher than that for pigeon isolates.